This invention relates generally to optical resonance analysis systems, specifically to certain sensor design aspects and to analysis systems comprising illumination and detection systems that utilize those sensors for analysis
Because of the recent surge in applications, sensor based instruments are becoming very popular. This growth in applications has been primarily spurred by the biotechnology and the pharmaceutical industries especially from the enormous influx of information from the Human Genome Program. This drove of information has resulted in a corresponding spawning of new industries. Some of the newest, rapidly growing industries are: proteomics, where proteins, function and genomics come together; and pharmacokinetics where researchers attempt to find products of combinatorial synthesis that have binding properties to unique sites such as receptors, that typically result in a biological altering event. Both technologies rely on assays to be robust and process thousands of samples/day. It is obvious that handling this amount of material at these speeds would benefit from automated processes and miniaturization. One very popular application such as monitoring DNA/DNA, DNA/RNA, RNA/RNA hybridization has always been important, but as genes are discovered and associated with disease states, genetic analysis in diagnostics requiring hybridization assays becomes a necessity. However, to obtain the information to determine the genetically relevant data, thousands of tests need to be run on one sample if conventional technologies are used. New developments in sensor technology can reduce this analysis time from weeks to hours.
Sensors can be described as being composed of two parts; the transducer and the active site. The transducer is defined as the part of the device that is capable of reporting change in its environment. Transducers can operate in several different modes but the most common are optical based devices. Examples of optical based transducers include surface plasmon resonance (SPR) devices and planar waveguide devices and grating coupled waveguide devices. These types of sensors are described in U.S. Pat. Nos. 4,882,288, 4931,384, 4,992,385, and 5,118,608 all incorporated by reference. The sensor may consist of a single analysis site, a one dimensional or linear array of analysis sites or a two dimensional array of analysis sites.
Surface plasmon, which exists at the boundary between metal and dielectric, represents a mode of surface charge vibrations. The surface charge vibration is the vibration of the electrons on the metal surface generated by exterior light, these electrons behaving like free electrons. The surface plasmon wave extends into space or dielectrics as an evanescent wave and travels along the surface. The plasmon field satisfies the Maxwell equations and boundary conditions for p-polarized radiation. This boundary condition requires that the dielectric constants of metal and dielectrics have opposite sign. Since the common dielectric compound has a positive dielectric constant, the plasmon exists in the frequency region of the metal where the dielectric constant is negative. This situation happens at a frequency of the exterior light and lower frequencies, in which the real part of the refractive index of the metal is equal to or smaller than its imaginary part. For instance, for metals such as Gold, Silver or Aluminum, this frequency, the plasmon frequency, is about 5, 4 or 15 eV, respectively, resulting in a plasmon wave being available in a frequency range covering WV, Visible and Infrared regions. In this frequency range, since the wave vector of the surface plasmon is larger than that of the exterior light, the exterior light cannot interact directly with surface plasmon.
Utilization of the surface plasmon becomes possible when the exterior light wave is coupled with the surface plasmon by means of a grating or prism. These optical components provide an additional wave vector component to the exterior light, enabling energy exchange between the exterior light and the surface plasmon. The plasmon on the metal grating can interact with the exterior light by picking up an additional transverse momentum defined by the period of the structure.
On the other hand (as in the back illuminated Kretchman design), attenuated total reflection in a high refractive index material such as a prism provides additional transverse momentum so that the exterior wave has a wave vector larger than the vacuum wave vector, and the wave vector in the prism is large enough to match to the plasmon wave vector.
The prism method has been frequently utilized to determine optical constants of metals, because the resonance condition changes by the change in the refractive index. As gratings play an important role in promoting the surface plasmon, this in turn means that the surface plasmon causes some anomalies to grating performance. Because of the phenomena, theory of surface plasmons was also developed by grating scientists.
The SPR type device basically measures refractive index changes in a thin 1 xcexcm evanescent field zone at its surface. The active surface defines the application and the specificity of the transducer. Various types of surface modifications can be used, for example, polymer coated transducers can be used to measure volatile organic compounds, bound proteins can be used to look for trace amounts of pesticides or other interactive molecules, DNA can be used to look for the presence of complementary DNA or even compounds that bind unique DNA sites. Specific sensors can be obtained by generating arrays of specific DNA sequences that hybridize the sample DNA. This technique is commonly referred to as array hybridization.
This type of sensor can operate in a gas or a liquid environment, as long as its performance is not degraded. Temperature range is selected by the application and should be controlled to better than 0.1xc2x0 C. for maximum sensitivity measurements.
Arrays have been built using fluorescence as the reporter but technologies such as SPR may be used resulting in reduced hardware cost, and greater generality. The use of SPR is especially appropriate in monitoring the binding of combinatorial products because these products will not all have labels or properties such as fluorescence that one could monitor. An extension of surface plasmon resonance is the ability to combine this technique with others such as mass spectrometry. An example would be if a signal is detected on the SPR sensors indicating binding, a second technique could be used to identify the bound material.
Surface Plasmon Resonance
The propagation of electromagnetic wave is expressed in terms of the waves equation as
E(x,t)=E0exp i(Kxxxe2x88x92xcfx89t)xe2x80x83xe2x80x83(1)
where Kx and D represent the wave vector in the x-direction and the angular frequency of the wave, respectively. The terms, x and t are distance and time, respectively. The plasmon wave vector is given by
Kx=2xcfx80v[xcex50+xcex51]xc2xd=(xcfx89/c)[xcex50xcex51/(xcex50+xcex51]xc2xd=(2xcfx80/xcex)[xcex50xcex51/(xcex50+xcex51]xc2xd.xe2x80x83xe2x80x83(2)
where xcex50 and xcex51 are dielectric constants of dielectric compound and metal and xcex is the wavelength of the exterior light. Twice the imaginary part of Kx, 2Kxi, determines the distance the plasmon electric field decays to 1/e along the metal surface.
Gratings provide the standing wave vector parallel to the boundary depending on the groove space and order of the grating. Thus, resonance absorption occurs when the exterior light wave vector component in the boundary plus the grating vector equal to the plasmon vector as given by
xe2x80x83(xcfx89/c)sin xcex8+2xcfx80m/a=(xcfx89/c)[xcex50xcex51/(xcex50+xcex51)]xc2xdxe2x80x83xe2x80x83(3)
where a and m are the groove spacing and the order of grating. Term xcex8 is the incident angle of the exterior light.
Resonance Width
For a given metal/dielectric boundary, the SPR wavevector Kx corresponding to a given frequency xcfx89 can be estimated as given in eq (1).
We define ksp as the real part of the center wavevector of the plasmon. The Lorentzian full width at half maximum, in the absence of radiative coupling, is given by twice the imaginary part of the wavevector, ki.
xcex94kFWHM=2 kixe2x80x83xe2x80x83(4)
The HWHM xcex94kxc2xd is obviously half of this value.:
xcex94kxc2xd=kixe2x80x83xe2x80x83(5)
Addition of radiative coupling or other losses can only increase the SPR linewidth. The question we seek to answer is the following: What is the non-radiative SPR width observed in terms of input wavelength xcex or angle xcex8, for both the grating coupling and prism coupling (Otto or Kretchman) cases.
Grating Coupling
ksp=(2xcfx80/xcex) sin xcex8+(2xcfx80m/a)xe2x80x83xe2x80x83(6)
where xcex is the vacuum wavelength, xcex8 is the input angle in air (not in the sample medium), m is the integer grating coupling order, and a is the grating groove pitch.
Prism Coupling:
ksp=(2xcfx80n/xcex) sin xcex8Pxe2x80x83xe2x80x83(7)
where n is the refractive index of the coupling prism and xcex8P is the input coupling angle within the prism medium.
In the case where the wavelength is fixed and the angle varies, since xcex is unchanged and monochromatic, the plasmon itself is unaffected by the angle change. Only the efficiency of in-coupling is affected. Imagine that the angle is initially set at xcex8 such that the plasmon is maximally excited. Now we shift the input angle to xcex8xe2x80x2 such that the excitation is reduced by 50%, to one of the half intensity points. Then we have simply
dxcex8xc2xd=xcex8xe2x80x2xe2x88x92xcex8=xcex94kxc2xd[∂xcex8/∂k]xe2x80x83xe2x80x83(8)
For the grating case, Eqn 6 gives
∂xcex8/∂k=xcex/{2xcfx80 cos xcex8}xe2x80x83xe2x80x83(9)
so that the in-air half-angle is
dxcex8xc2xd=xcex94kxc2xdxcex/(2xcfx80 cos xcex8)xe2x80x83xe2x80x83(10)
For the prism case, Eqn 7 gives
∂xcex8P/∂k=xcex/{2xcfx80n cos xcex8P}xe2x80x83xe2x80x83(11)
so that the in-prism half-angle is
dxcex8xc2xd(Prism)=xcex94kxc2xdxcex/(2xcfx80n cos xcex8)xe2x80x83xe2x80x83(12)
If the prism is beveled to allow near-normal incidence coupling from air into the prism, then according to Snell""s law the differential angle in air is n times that inside the glass. The net result for the prism case is that the in-air half-angle is
dxcex8xc2xd(Air)=xcex94kxc2xdxcex/(2xcfx80 cos xcex8P),xe2x80x83xe2x80x83(13)
a result nearly identical to the grating result (10).
In other words, to the extent that the nominal incoupling angle in air for the grating case is similar to the incoupling angle in glass for the prism case, the in-air angular resonance widths are nearly the same. The full FWHM angular width in air, for either case, is found from doubling (10) or (13) to be
xe2x80x83xcex94xcex8FWHM=2 kixcex/(2xcfx80 cos xcex8)xe2x80x83xe2x80x83(14)
In the case where the angle is fixed and the wavelength varies, since xcfx89 varies when we change xcex, the plasmon itself changes as we vat, the input wavelength. At the same time, the coupling conditions also change so the new plasmon is not being excited on-resonance. Both effects need to be taken properly into account.
Assume that we start, as before, with xcex and xcex8 chosen so that we are tuned to the SPR peak initially, which has wavevector ksp. Now we change the wavelength to xcexxe2x80x2 so as to attempt to reach the half intensity point. That is, we seek to have
xcexxe2x80x2=xcex+xcex94xcexxc2xdxe2x80x83xe2x80x83(15)
As we do this, the plasmon wavevector must change to kxe2x80x2sp such that
kxe2x80x2sp32 ksp+(∂k/∂xcex) (xcexxe2x80x2xe2x88x92xcex)xe2x80x83xe2x80x83(16)
Here, the partial ∂k/∂xcex is calculated numerically from (1) using tabulated dielectric constant data for the materials forming the SPR device. Note that in general, it is negative.
At the same time the wavevector kL being launched is given by Eqn (6) or (7), depending on what kind of coupler we are using.
For the grating case, we launch at wavevector                                                                         k                L                            =                              xe2x80x83                            ⁢                                                                    (                                          2                      ⁢                                              π                        /                                                  λ                          xe2x80x2                                                                                      )                                    ⁢                  sin                  ⁢                                      xe2x80x83                                    ⁢                  θ                                +                                  (                                      2                    ⁢                    π                    ⁢                                          xe2x80x83                                        ⁢                                          m                      /                      a                                                        )                                                                                                        =                              xe2x80x83                            ⁢                                                k                  sp                                +                                  2                  ⁢                  π                  ⁢                                      xe2x80x83                                    ⁢                  sin                  ⁢                                      xe2x80x83                                    ⁢                                      θ                    ⁡                                          [                                                                        1                          /                                                      λ                            xe2x80x2                                                                          -                                                  1                          /                          λ                                                                    ]                                                                                                                                                              ≅                                  xe2x80x83                                ⁢                                                      k                    sp                                    -                                      2                    ⁢                    π                    ⁢                                          xe2x80x83                                        ⁢                    sin                    ⁢                                          xe2x80x83                                        ⁢                                          θ                      ⁡                                              [                                                                              (                                                                                          λ                                xe2x80x2                                                            -                              λ                                                        )                                                    /                                                      λ                            xe2x80x2                                                                          ]                                                                                                        ,                                                          (        17        )            
whereas for the prism case                                                                         k                L                            =                              xe2x80x83                            ⁢                                                                    (                                          2                      ⁢                      π                      ⁢                                              xe2x80x83                                            ⁢                                              n                        /                                                  λ                          xe2x80x2                                                                                      )                                    ⁢                  sin                  ⁢                                      xe2x80x83                                    ⁢                  θ                                =                                                                            [                                              λ                        /                                                  λ                          xe2x80x2                                                                    ]                                        ⁢                                          k                      sp                                                        =                                                            [                                              λ                        /                                                  (                                                      λ                            +                            Δλ                                                    )                                                                    ]                                        ⁢                                          k                      sp                                                                                                                                                              ≅                                  xe2x80x83                                ⁢                                                      [                                                                  (                                                  λ                          -                          Δλ                                                )                                            /                      λ                                        ]                                    ⁢                                      k                    sp                                                              =                                                k                  sp                                -                                                      [                                          Δλ                      /                      λ                                        ]                                    ⁢                                      k                    sp                                                                                                          (        18        )            
To reach the half intensity point by tuning xcex, we require that the mismatch between the launch wavevector kL and the modified plasmon wavevector kspxe2x80x2 be exactly xcex94kxc2xd=ki:
xe2x80x83kLxe2x88x92ksp=xc2x1kixe2x80x83xe2x80x83(19)
Combining (16) and (17), Eqn 19 becomes, for the grating case,
xe2x88x922xcfx80sin xcex8[(xcexxe2x80x2xe2x88x92xcex)/xcex2]xe2x88x92(∂k/∂xcex)(xcexxe2x80x2xe2x88x92xcex)=xc2x1kixe2x80x83xe2x80x83(19)
or                                           Δ            ⁢                          xe2x80x83                        ⁢                          λ                              1                /                2                                              ≡                                    λ              xe2x80x2                        -            λ                          =                  -                                    ±                              k                i                                                                    2                ⁢                π                ⁢                                  xe2x80x83                                ⁢                sin                ⁢                                  xe2x80x83                                ⁢                                  θ                  /                                      λ                    2                                                              +                                                ∂                  k                                /                                  ∂                  λ                                                                                        (        20        )            
For the prism case, Eqns (16) and (18) lead in a similar fashion to
xe2x88x92ksp[(xcexxe2x80x2xe2x88x92xcex)/xcexxe2x88x92(∂k/∂xcex)(xcexxe2x80x2xe2x88x92xcex)=xc2x1ki
or                                           Δ            ⁢                          xe2x80x83                        ⁢                          λ                              1                /                2                                              ≡                                    λ              xe2x80x2                        -            λ                          =                  -                                    ±                              k                i                                                                                      k                  sp                                /                λ                            +                                                ∂                  k                                /                                  ∂                  λ                                                                                        (        21        )            
Note that the HWHM in xcex given by (20) and (21) for grating and prism coupling respectively give quite different results. In general, the width will be wider for prism coupling, since the two terms in the denominator have opposite signs, for example the first term is positive and the second term is negative, and similar magnitudes, tending to reduce the denominator and hence increase the quotient.
Note also that the FWHM resonance widths are double the HWHM values of (20) and (21):
xcex94xcexFWHM=2 xcex94xcexxc2xd
Waveguide sensors consist of one or more layers of dielectric materials coated with a thin film of material of higher index of refraction. The waveguide sensor responds to: changes in the refractive index nC of the cover medium C; adsorption of molecules out of a gaseous or liquid phase cover, to form a surface layer of thickness dr and refraction index nF. and, if used as dispersing element (Propagation angle in the waveguide depends on the wavelength), it can record the absorption spectrum of molecules on the surface. The sensitivity can be expressed as the change in the effective index of refraction N (of a guided mode TE or TM) in cases 1-3. In the case of absorption measurements (and using the guide as a dispersive element) the sensitivity is determined by the minimum detectable absorption.
This type of sensor can operate in a gas or a liquid environment, as long as its performance is not degraded. Temperature range is selected by the application and should be controlled to better than 0.1xc2x0 C. for maximum sensitivity measurements. Substrates include sapphire, ITO, fused silica, glass (Pyrex, Quartz,), plastic, Teflon, metal, and semiconductor materials (Silicon). Waveguide films include SiO2, SiO2xe2x80x94TiO2, TiO2, Si3N4, lithium niobate, lithium tantalate, tantalum pentoxide, niobium pentoxide, GaAs, GaAlAs, GaAsP, GaInAs, and polymers (polystyrene). Waveguide film thickness is usually in the range of 100-200 nm. Example ranges of indexes of refraction include from 1.4-2.1. Chemoselective coatings can be placed on the waveguide film surface. Light coupling into the waveguide can be achieved by using surface relief gratings or prisms.
A way to measure the changes in the effective index is by the change in the angle at which the mode exits from the waveguide. This can be done by an array detector, which at the same time can measure the intensity at each wavelength. In that case the time of measurement is typically 100-200 xcexcsec.
Associated system components usually include gratings (to match the Dangle dispersion curve of the waveguide), mirrors, lenses, polarizers, white light sources, and array detectors.
Multiple assays and analytes are possible as long as the waveguide can be spotted with different chemistries, the incoming light is split into multiple collimated light beams, there is no mixing of the light beams inside the guide, and the detection can be done simultaneously for all assays/analytes.
Possible applications are analytical chemistry, humidity and gas sensing, PH measurement, bio- and immuno-sensor applications, molecular recognition in biology, signaling transduction between and within cells, affinity of biotinylated molecules (bovine serum albumin) to Avidin or strept-Avidin, antigen-antibody interactions (immunobinding of rabbit/goat anti-h-IgG antibody to the human immunoglobulin h-IgG antigen) etc. The grating coupled waveguide sensor can measure the number, size and shape of living cells growing on its surface, in real time and non-invasively. Applications include toxicology and cancer research, pharmacologyxe2x80x94drug determination. A waveguide supported lipid bilayer is the closest to real cell membrane simulation, and can be used for drug screening as well as blood-brain barriers. Waveguides can be used to analyze properties of bilayer lipid membranes (BLM) and other thin films, to measure protein-BLM interaction, and the thickness, density, anisotropy, and the reaction of thin films to perturbations in time. Other applications include using long DNA molecules as a surface coat to measure hybridization and protein binding, molecular self-assembly, nanoscience, and analysis of association and dissociation kinetics.
Grating couplers are used for efficient coupling of light into or out of a waveguide that consists of one or more layers of dielectric materials. At the same time they can be used for measuring the effective index of refraction N of all possible TE and TM modes. The primary sensor effect is a change xcex94N in the effective refractive index N of the guided modes induced by the adsorption or binding of molecules from a sample on the waveguide surface. From xcex94N it is possible to calculate the refractive index, thickness and surface coverage of the adsorbed or bound adlayers. Provided that thin monomode waveguide film F with a large difference nF-nS between the refractive indexes of film F and substrate S are used, integrated optics guarantees high sensor sensitivities (sub-monomolecular adsorbed layer). xcex94N is measured only in the grating region which is where the sample should be placed. With optimal grating design a coupling efficiency of the order of 45-90% can be achieved.
A grating coupler can operate in a gas or a liquid environment, as long as its perfomance is not degraded. Temperature range is selected by the application and should be controlled to better than 0.1xc2x0 C. for maximum sensitivity measurements. Substrates include sapphire, ITO, fused silica, glass (Pyrex, Quartz,), plastic, teflon, metal, and semiconductor materials (Silicon). Waveguide films include Sio2, SiO2xe2x80x94TiO2, TiO2, Si3N4, lithium niobate, lithium tantalate, tantalum pentoxide, niobium pentoxide, GaAs, GaAIAs, GaAsP, GalnAs, and polymers (polystyrene). Waveguide film thickness can be in the range of 100-200 nm and indexes of refraction can be in the range of 1.4-2.1. Gratings can be made by embossing, ion-implantation and photoresist techniques, on the substrate or in the waveguide film. Typical numbers are 1200-2400 lines/mm, 2xc3x9716 mm in size and 1:1 aspect ratio (20 nm features). Chemoselective coatings can be placed on the waveguide film surface.
For incoupling gratings mechanical angle scanning measurement time is typically 2-3 sec. If an array of sources is used in conjunction with a lens this time is shortened. For an outcoupling grating and a position sensitive detector (no moving parts), it is typically 100-200 xcexcsec.
Associated system components usually include optics, mirrors, lenses, polarizers, light sources, light source arrays, laser sources, single or position sensitive detectors, rotation stages, and stepper motors
Multiple assays and analytes are possible as long as the waveguide can be spotted with different chemistries, the incoming light is split into multiple collimated light beams, there is no mixing of the light beams inside the guide, and the detection can be done simultaneously for all assays or analytes.
Applications include analytical chemistry, humidity and gas sensing, PH measurement, bio- and immuno-sensor applications, molecular recognition in biology, signaling transduction between and within cells, affinity of biotinylated molecules (bovine serum albumin) to Avidin or strept-Avidin), antigen-antibody interactions (immunobinding of rabbit/goat anti-h-IgG antibody to the human immunoglobulin h-IgG antigen) etc.
Analysis systems utilizing these types of optical resonance devices (SPR and waveguides) typically include an illumination system having the capability to project light at various frequencies or angles onto the resonance device and a detection system for detecting the corresponding resonance peaks.
The illumination systems are typically composed of a light source, a means for causing the light source to impinge on the sensor at different angles or at different frequencies, and optics to facilitate imaging the source onto the sensor. The choice of light source is based on the wavelength region required and the etendue (solid angle X photon flux) of the optical system. There are a large variety of broadband or monochromatic sources to choose from, such as: incandescent, LED""s, super luminescent diodes, lasers (fixed and tunable, diode, solid state, gas), gas discharge lamps (line and continuum), with or without filters. Wavelength scanning is usually accomplished by coupling the source with filter wheels, scanning monochrometers or acousto-optical tunable filters, or in the case of a laser source by using a tunable diode laser. Angle scanning is usually accomplished by mechanically positioning the source at a series of angles with relationship to the sensor. In addition the source must be oriented and focused so that it optimally projects onto the sensor.
Light rays from the illumination system are reflected from the sensor with their angle of reflection equal to their angle of incidence. Thus the rays will typically span a small range of angles in the perpendicular plane. The detector is typically positioned to optimally receive the rays coming from the sensor. Other important detector considerations are resolution, pixel size, number of pixels, the algorithms that will be used for analyzing the resonance wavelengths or angles, and the chemistry occurring on the detector.
In resonance measurement, a peak or a dip is obtained over a sometime sloped baseline. When the measurement is performed on another sample at a different concentration, this peak or dip will shift depending on the change in refractive index corresponding to concentration difference between the two samples. The concentration can then be predicted using a calibration model relating the peak or dip shift to concentration.
This invention is directed toward an analysis system, comprising an illumination and detection system, which utilizes a surface plasmon device or a waveguide as a sensor.
The invention is further directed toward an illumination system utilizing an array of light sources to facilitate angle or wavelength scanning.
The invention is further directed toward a means for providing independent axial and rotational positioning of the array of light sources.
The invention is further directed toward utilizing a diffraction grating or a diffractive optical element as a pre-dispersive or post-dispersive element in the analysis system allowing the use of low cost, broad band light sources.
The invention is further directed toward an anamorphic optical design that allows the analysis system to generate and detect resonances, from a one dimensional array of sites on the same substrate, simultaneously.
The invention further comprises a method for predicting concentrations using a calibration model relating the resonance peak shift to concentration.